Isolation of human fibroadipogenic progenitors and satellite cells from frozen muscle biopsies.

Skeletal muscle contains multiple cell types that work together to maintain tissue homeostasis. Among these, satellite cells (SC) and fibroadipogenic progenitors cells (FAPs) are the two main stem cell pools. Studies of these cells using animal models have shown the importance of interactions between these cells in repair of healthy muscle, and degeneration of dystrophic muscle. Due to the unavailability of fresh patient muscle biopsies, similar analysis of interactions between human FAPs and SCs is limited especially among the muscular dystrophy patients. To address this issue here we describe a method that allows the use of frozen human skeletal muscle biopsies to simultaneously isolate and grow SCs and FAPs from healthy or dystrophic patients. We show that while the purified SCs differentiate into mature myotubes, purified FAPs can differentiate into adipocytes or fibroblasts demonstrating their multipotency. We find that these FAPs can be immortalized and the immortalized FAPs (iFAPs) retain their multipotency. These approaches open the door for carrying out personalized analysis of patient FAPs and interactions with the SCs that lead to muscle loss.

Optimal Preclinical Conditions for Using Adult Human Multipotent Neural Cells in the Treatment of Spinal Cord Injury.

Stem cell-based therapeutics are amongst the most promising next-generation therapeutic approaches for the treatment of spinal cord injury (SCI), as they may promote the repair or regeneration of damaged spinal cord tissues. However, preclinical optimization should be performed before clinical application to guarantee safety and therapeutic effect. Here, we investigated the optimal injection route and dose for adult human multipotent neural cells (ahMNCs) from patients with hemorrhagic stroke using an SCI animal model. ahMNCs demonstrate several characteristics associated with neural stem cells (NSCs), including the expression of NSC-specific markers, self-renewal, and multi neural cell lineage differentiation potential. When ahMNCs were transplanted into the lateral ventricle of the SCI animal model, they specifically migrated within 24 h of injection to the damaged spinal cord, where they survived for at least 5 weeks after injection. Although ahMNC transplantation promoted significant locomotor recovery, the injection dose was shown to influence treatment outcomes, with a 1 × 106 (medium) dose of ahMNCs producing significantly better functional recovery than a 3 × 105 (low) dose. There was no significant gain in effect with the 3 × 106 ahMNCs dose. Histological analysis suggested that ahMNCs exert their effects by modulating glial scar formation, neuroprotection, and/or angiogenesis. These data indicate that ahMNCs from patients with hemorrhagic stroke could be used to develop stem cell therapies for SCI and that the indirect injection route could be clinically relevant. Moreover, the optimal transplantation dose of ahMNCs defined in this preclinical study might be helpful in calculating its optimal injection dose for patients with SCI in the future.

Graft-versus-host disease depletes plasmacytoid dendritic cell progenitors to impair tolerance induction.

Graft-versus-host disease (GVHD) causes failed reconstitution of donor plasmacytoid dendritic cells (pDCs) that are critical for immune protection and tolerance. We used both murine and human systems to uncover the mechanisms whereby GVHD induces donor pDC defects. GVHD depleted Flt3-expressing donor multipotent progenitors (MPPs) that sustained pDCs, leading to impaired generation of pDCs. MPP loss was associated with decreased amounts of MPP-producing hematopoietic stem cells (HSCs) and oxidative stress-induced death of proliferating MPPs. Additionally, alloreactive T cells produced GM-CSF to inhibit MPP expression of Tcf4, the transcription factor essential for pDC development, subverting MPP production of pDCs. GM-CSF did not affect the maturation of pDC precursors. Notably, enhanced recovery of donor pDCs upon adoptive transfer early after allogeneic HSC transplantation repressed GVHD and restored the de novo generation of donor pDCs in recipient mice. pDCs suppressed the proliferation and expansion of activated autologous T cells via a type I IFN signaling-dependent mechanism. They also produced PD-L1 and LILRB4 to inhibit T cell production of IFN-γ. We thus demonstrate that GVHD impairs the reconstitution of tolerogenic donor pDCs by depleting DC progenitors rather than by preventing pDC maturation. MPPs are an important target to effectively bolster pDC reconstitution for controlling GVHD.

Moderate Alcohol Consumption Targets S100ß+ Vascular Stem Cells and Attenuates Injury-Induced Neointimal Hyperplasia.

BACKGROUND: Stem cells present in the vessel wall may be triggered in response to injurious stimuli to undergo differentiation and contribute to vascular disease development. Our aim was to determine the effect of moderate alcohol (EtOH) exposure on the expansion and differentiation of S100 calcium-binding protein B positive (S100ß+ ) resident vascular stem cells and their contribution to pathologic vessel remodeling in a mouse model of arteriosclerosis. METHODS AND RESULTS: Lineage tracing analysis of S100ß+ cells was performed in male and female S100ß-eGFP/Cre/ERT2-dTomato transgenic mice treated daily with or without EtOH by oral gavage (peak BAC: 15 mM or 0.07%) following left common carotid artery ligation for 14 days. Carotid arteries (ligated or sham-operated) were harvested for morphological analysis and confocal assessment of fluorescent-tagged S100 ß + cells in FFPE carotid cross sections. Ligation-induced carotid remodeling was more robust in males than in females. EtOH-gavaged mice had less adventitial thickening and markedly reduced neointimal formation compared to controls, with a more pronounced inhibitory effect in males compared to females. There was significant expansion of S100ß+ -marked cells in vessels postligation, primarily in the neointimal compartment. EtOH treatment reduced the fraction of S100ß+ cells in carotid cross sections, concomitant with attenuated remodeling. In vitro, EtOH attenuated Sonic Hedgehog-stimulated myogenic differentiation (as evidenced by reduced calponin and myosin heavy chain expression) of isolated murine S100ß+ vascular stem cells. CONCLUSIONS: These data highlight resident vascular S100ß+ stem cells as a novel target population for alcohol and suggest that regulation of these progenitors in adult arteries, particularly in males, may be an important mechanism contributing to the antiatherogenic effects of moderate alcohol consumption.

Ischemia-Induced Multipotent Stem Cells Isolated from Stroke Patients Exhibit Higher Neurogenic Differentiation Potential than Bone Marrow-Derived Mesenchymal Stem Cells.

Perivascular areas of the brain harbor multipotent stem cells. We recently demonstrated that after a stroke, brain pericytes exhibit features of multipotent stem cells. Moreover, these ischemia-induced multipotent stem cells (iSCs) are present within ischemic areas of the brain of patients diagnosed with stroke. Although increasing evidence shows that iSCs have traits similar to those of mesenchymal stem cells (MSCs), the phenotypic similarities and differences between iSCs and MSCs remain unclear. In this study, we used iSCs extracted from stroke patients (h-iSCs) and compared their neurogenic potential with that of human MSCs (h-MSCs) in vitro. Microarray analysis, fluorescence-activated cell sorting, immunohistochemistry, and multielectrode array were performed to compare the characteristics of h-iSCs and h-MSCs. Although h-iSCs and h-MSCs had similar gene expression profiles, the percentage expressing the neural stem/progenitor cell marker nestin was significantly higher in h-iSCs than in h-MSCs. Consistent with these findings, h-iSCs, but not h-MSCs, differentiated into electrophysiologically functional neurons. In contrast, although both h-iSCs and h-MSCs were able to differentiate into several mesodermal lineages, including adipocytes, osteocytes, and chondrocytes, the potential of h-iSCs to differentiate into adipocytes and osteocytes was relatively low. These results suggest that compared with h-MSCs, h-iSCs predominantly exhibit neural rather than mesenchymal lineages. In addition, these results indicate that h-iSCs have the potential to repair the injured brain of patients with stroke by directly differentiating into neuronal lineages.

N-acetylcysteine prevents oxidized low-density lipoprotein-induced reduction of MG53 and enhances MG53 protective effect on bone marrow stem cells.

MG53 is an important membrane repair protein and partially protects bone marrow multipotent adult progenitor cells (MAPCs) against oxidized low-density lipoprotein (ox-LDL). The present study was to test the hypothesis that the limited protective effect of MG53 on MAPCs was due to ox-LDL-induced reduction of MG53. MAPCs were cultured with and without ox-LDL (0-20 µg/mL) for up to 48 hours with or without MG53 and antioxidant N-acetylcysteine (NAC). Serum MG53 level was measured in ox-LDL-treated mice with or without NAC treatment. Ox-LDL induced significant membrane damage and substantially impaired MAPC survival with selective inhibition of Akt phosphorylation. NAC treatment effectively prevented ox-LDL-induced reduction of Akt phosphorylation without protecting MAPCs against ox-LDL. While having no effect on Akt phosphorylation, MG53 significantly decreased ox-LDL-induced membrane damage and partially improved the survival, proliferation and apoptosis of MAPCs in vitro. Ox-LDL significantly decreased MG53 level in vitro and serum MG53 level in vivo without changing MG53 clearance. NAC treatment prevented ox-LDL-induced MG53 reduction both in vitro and in vivo. Combined NAC and MG53 treatment significantly improved MAPC survival against ox-LDL. These data suggested that NAC enhanced the protective effect of MG53 on MAPCs against ox-LDL through preventing ox-LDL-induced reduction of MG53.

Characterization of mesoangioblast cell fate and improved promyogenic potential of a satellite cell-like subpopulation upon transplantation in dystrophic murine muscles.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease caused by the lack of dystrophin in muscle fibers that is currently without curative treatment. Mesoangioblasts (MABs) are multipotent progenitor cells that can differentiate to a myogenic lineage and that can be used to express Dystrophin upon transplantation into muscles, in autologous gene therapy approaches. However, their fate in the muscle environment remains poorly characterized. Here, we investigated the differentiation fate of MABs following their transplantation in DMD murine muscles using a mass cytometry strategy. This allowed the identification and isolation of a fraction of MAB-derived cells presenting common properties with satellite muscle stem cells. This analysis also indicated that most cells did not undergo a myogenic differentiation path once in the muscle environment, limiting their capacity to restore dystrophin expression in transplanted muscles. We therefore assessed whether MAB treatment with cytokines and growth factors prior to engraftment may improve their myogenic fate. We identified a combination of such signals that ameliorates MABs capacity to undergo myogenic differentiation in vivo and to restore dystrophin expression upon engraftment in myopathic murine muscles.

Derivation of neural stem cells from human teratomas.

Human teratoma is a germ cell tumor that contains normal tissues (e.g., hair, skin or cartilage) differentiated from embryonal germ layers. Because of the feature of this tumor, we hypothesized that human teratomas contain multipotent stem cells that can develop into various non-cancerous normal tissues. In this study, we cultured neurospheres originally derived from a human infantile teratoma tissue, and the sphere cells were found to possess the characteristics of neural stem cells. Tumor tissues were obtained from an infantile immature teratoma at the time of surgical resection. In the primary cell culture, colonies were formed in two weeks and were individually cultured in serum-free conditioned neural stem cell medium (NSC medium). Colonies changed into spheres and grew in smooth round forms, or attached to the bottom of the dishes and extended processes and filaments around. Sphere cells were dissociated into single cells, and new spheres (secondary spheres) were formed in NSC medium. Cell differentiation was induced by culturing cells in serum-containing medium (differentiation medium), as cells spread and attached to the bottom of dishes and changed form. The expression of Nestin, Sox2, CXCR4, and (stem cell markers), ß3-tubulin (a neural marker) GFAP (a glial marker) CNPase, SOX10 (oligodendrocyte markers) and NF-L in cells was analyzed by immunofluorescence and a Q-PCR. Nestin, SOX2, CXCR4 were abundant in both primary and secondary spheres. Neural and glial markers (ß3-tubulin and GFAP, respectively) were increased in cells cultured in differentiation medium while stem cell markers were diminished. The oligodendrocyte markers SOX10 and CNPase were also found in both spheres and differentiated cells. In conclusion, spheres with the characteristics of neural stem cells were obtained from the primary culture of a human infantile teratoma. These spheres are considered to have the potential to undergo a natural course of neural development in humans.

Niche stiffness underlies the ageing of central nervous system progenitor cells.

Ageing causes a decline in tissue regeneration owing to a loss of function of adult stem cell and progenitor cell populations1. One example is the deterioration of the regenerative capacity of the widespread and abundant population of central nervous system (CNS) multipotent stem cells known as oligodendrocyte progenitor cells (OPCs)2. A relatively overlooked potential source of this loss of function is the stem cell 'niche'-a set of cell-extrinsic cues that include chemical and mechanical signals3,4. Here we show that the OPC microenvironment stiffens with age, and that this mechanical change is sufficient to cause age-related loss of function of OPCs. Using biological and synthetic scaffolds to mimic the stiffness of young brains, we find that isolated aged OPCs cultured on these scaffolds are molecularly and functionally rejuvenated. When we disrupt mechanical signalling, the proliferation and differentiation rates of OPCs are increased. We identify the mechanoresponsive ion channel PIEZO1 as a key mediator of OPC mechanical signalling. Inhibiting PIEZO1 overrides mechanical signals in vivo and allows OPCs to maintain activity in the ageing CNS. We also show that PIEZO1 is important in regulating cell number during CNS development. Thus we show that tissue stiffness is a crucial regulator of ageing in OPCs, and provide insights into how the function of adult stem and progenitor cells changes with age. Our findings could be important not only for the development of regenerative therapies, but also for understanding the ageing process itself.

CDKN2B upregulation prevents teratoma formation in multipotent fibromodulin-reprogrammed cells.

Tumorigenicity is a well-documented risk to overcome for pluripotent or multipotent cell applications in regenerative medicine. To address the emerging demand for safe cell sources in tissue regeneration, we established a novel, protein-based reprogramming method that does not require genome integration or oncogene activation to yield multipotent fibromodulin (FMOD)-reprogrammed (FReP) cells from dermal fibroblasts. When compared with induced pluripotent stem cells (iPSCs), FReP cells exhibited a superior capability for bone and skeletal muscle regeneration with markedly less tumorigenic risk. Moreover, we showed that the decreased tumorigenicity of FReP cells was directly related to an upregulation of cyclin-dependent kinase inhibitor 2B (CDKN2B) expression during the FMOD reprogramming process. Indeed, sustained suppression of CDKN2B resulted in tumorigenic, pluripotent FReP cells that formed teratomas in vivo that were indistinguishable from iPSC-derived teratomas. These results highlight the pivotal role of CDKN2B in cell fate determination and tumorigenic regulation and reveal an alternative pluripotent/multipotent cell reprogramming strategy that solely uses FMOD protein.

Muscle injury-induced hypoxia alters the proliferation and differentiation potentials of muscle resident stromal cells.

BACKGROUND: Trauma-induced heterotopic ossification (HO) is a complication that develops under three conditions: the presence of an osteogenic progenitor cell, an inducing factor, and a permissive environment. We previously showed that a mouse multipotent Sca1+ CD31- Lin- muscle resident stromal cell (mrSC) population is involved in the development of HO in the presence of inducing factors, members of the bone morphogenetic protein family. Interestingly, BMP9 unlike BMP2 causes HO only if the muscle is damaged by injection of cardiotoxin. Because acute trauma often results in blood vessel breakdown, we hypothesized that a hypoxic state in damaged muscles may foster mrSCs activation and proliferation and trigger differentiation toward an osteogenic lineage, thus promoting the development of HO. METHODS: Three- to - six-month-old male C57Bl/6 mice were used to induce muscle damage by injection of cardiotoxin intramuscularly into the tibialis anterior and gastrocnemius muscles. mrSCs were isolated from damaged (hypoxic state) and contralateral healthy muscles and counted, and their osteoblastic differentiation with or without BMP2 and BMP9 was determined by alkaline phosphatase activity measurement. The proliferation and differentiation of mrSCs isolated from healthy muscles was also studied in normoxic incubator and hypoxic conditions. The effect of hypoxia on BMP synthesis and Smad pathway activation was determined by qPCR and/or Western blot analyses. Differences between normally distributed groups were compared using a Student's paired t test or an unpaired t test. RESULTS: The hypoxic state of a severely damaged muscle increased the proliferation and osteogenic differentiation of mrSCs. mrSCs isolated from damaged muscles also displayed greater sensitivity to osteogenic signals, especially BMP9, than did mrSCs from a healthy muscle. In hypoxic conditions, mrSCs isolated from a control muscle were more proliferative and were more prone to osteogenic differentiation. Interestingly, Smad1/5/8 activation was detected in hypoxic conditions and was still present after 5 days, while Smad1/5/8 phosphorylation could not be detected after 3 h of normoxic incubator condition. BMP9 mRNA transcripts and protein levels were higher in mrSCs cultured in hypoxic conditions. Our results suggest that low-oxygen levels in damaged muscle influence mrSC behavior by facilitating their differentiation into osteoblasts. This effect may be mediated partly through the activation of the Smad pathway and the expression of osteoinductive growth factors such as BMP9 by mrSCs. CONCLUSION: Hypoxia should be considered a key factor in the microenvironment of damaged muscle that triggers HO.

HMGA1 negatively regulates NUMB expression at transcriptional and post transcriptional level in glioblastoma stem cells.

Glioblastoma (GBM) is a lethal, fast-growing brain cancer, affecting 2-3 per 100,000 adults per year. It arises from multipotent neural stem cells which have reduced their ability to divide asymmetrically and hence divide symmetrically, generating increasing number of cancer stem cells, fostering tumor growth. We have previously demonstrated that the architectural transcription factor HMGA1 is highly expressed in brain tumor stem cells (BTSCs) and that its silencing increases stem cell quiescence, reduces self-renewal and sphere-forming efficiency in serial passages, suggesting a shift from symmetric to asymmetric division. Since NUMB expression is fundamental for the fulfillment of asymmetric division in stem cells, and is lost or reduced in many tumors, including GBM, we have investigated the ability of HMGA1 to regulate NUMB expression. Here, we show that HMGA1 negatively regulates NUMB expression at transcriptional level, by binding its promoter and counteracting c/EBP-ß and at posttranscriptional level, by regulating the expression of MSI1 and of miR-146a. Finally, we report that HMGA1 knockdown-induced NUMB upregulation leads to the downregulation of the NOTCH1 pathway. Therefore, the data reported here indicate that HMGA1 negatively regulates NUMB expression in BTSCs, further supporting HMGA1 targeting as innovative and effective anti-cancer therapy.

p53-TP53-Induced Glycolysis Regulator Mediated Glycolytic Suppression Attenuates DNA Damage and Genomic Instability in Fanconi Anemia Hematopoietic Stem Cells.

Emerging evidence has shown that resting quiescent hematopoietic stem cells (HSCs) prefer to utilize anaerobic glycolysis rather than mitochondrial respiration for energy production. Compelling evidence has also revealed that altered metabolic energetics in HSCs underlies the onset of certain blood diseases; however, the mechanisms responsible for energetic reprogramming remain elusive. We recently found that Fanconi anemia (FA) HSCs in their resting state are more dependent on mitochondrial respiration for energy metabolism than on glycolysis. In the present study, we investigated the role of deficient glycolysis in FA HSC maintenance. We observed significantly reduced glucose consumption, lactate production, and ATP production in HSCs but not in the less primitive multipotent progenitors or restricted hematopoietic progenitors of Fanca-/- and Fancc-/- mice compared with that of wild-type mice, which was associated with an overactivated p53 and TP53-induced glycolysis regulator, the TIGAR-mediated metabolic axis. We utilized Fanca-/- HSCs deficient for p53 to show that the p53-TIGAR axis suppressed glycolysis in FA HSCs, leading to enhanced pentose phosphate pathway and cellular antioxidant function and, consequently, reduced DNA damage and attenuated HSC exhaustion. Furthermore, by using Fanca-/- HSCs carrying the separation-of-function mutant p53R172P transgene that selectively impairs the p53 function in apoptosis but not cell-cycle control, we demonstrated that the cell-cycle function of p53 was not required for glycolytic suppression in FA HSCs. Finally, ectopic expression of the glycolytic rate-limiting enzyme PFKFB3 specifically antagonized p53-TIGAR-mediated metabolic reprogramming in FA HSCs. Together, our results suggest that p53-TIGAR metabolic axis-mediated glycolytic suppression may play a compensatory role in attenuating DNA damage and proliferative exhaustion in FA HSCs. Stem Cells 2019;37:937-947.

Osteogenic differentiation of skeletal muscle progenitor cells is activated by the DNA damage response.

Heterotopic ossification (HO) is a pathological condition characterized by the deposition of mineralized tissue in ectopic locations such as the skeletal muscle. The precise cellular origin and molecular mechanisms underlying HO are still debated. In our study we focus on the differentiation of mesoangioblasts (MABs), a population of multipotent skeletal muscle precursors. High-content screening for small molecules that perturb MAB differentiation decisions identified Idoxuridine (IdU), an antiviral and radiotherapy adjuvant, as a molecule that promotes MAB osteogenic differentiation while inhibiting myogenesis. IdU-dependent osteogenesis does not rely on the canonical BMP-2/SMADs osteogenic pathway. At pro-osteogenic conditions IdU induces a mild DNA Damage Response (DDR) that activates ATM and p38 eventually promoting the phosphorylation of the osteogenesis master regulator RUNX2. By interfering with this pathway IdU-induced osteogenesis is severely impaired. Overall, our study suggests that induction of the DDR promotes osteogenesis in muscle resident MABs thereby offering a new mechanism that may be involved in the ectopic deposition of mineralized tissue in the muscle.

Isolation and Characterization of Cerebellum-Derived Stem Cells in Poststroke Human Brain.

There is compelling evidence that the mature central nervous system (CNS) harbors stem cell populations outside conventional neurogenic regions. We previously demonstrated that brain pericytes (PCs) in both mouse and human exhibit multipotency to differentiate into various neural lineages following cerebral ischemia. PCs are found throughout the CNS, including cerebellum, but it remains unclear whether cerebellar PCs also form ischemia-induced multipotent stem cells (iSCs). In this study, we demonstrate that putative iSCs can be isolated from poststroke human cerebellum (cerebellar iSCs [cl-iSCs]). These cl-iSCs exhibited multipotency and differentiated into electrophysiologically active neurons. Neurogenic potential was also confirmed in single-cell suspensions. DNA microarray analysis revealed highly similar gene expression patterns between PCs and cl-iSCs, suggesting PC origin. Global gene expression comparison with cerebral iSCs revealed general similarity, but cl-iSCs differentially expressed certain cerebellum-specific genes. Thus, putative iSCs are present in poststroke cerebellum and possess region-specific traits, suggesting potential capacity to regenerate functional cerebellar neurons following ischemic stroke.

Assays for functionally defined normal and malignant mammary stem cells.

The discovery of rare, heterogeneous self-renewing stem cells with shared developmental and molecular features within epithelial components of mammary gland and breast cancers has provided a conceptual framework to understand cellular composition of these tissues and mechanisms that control their number. These normal mammary epithelial stem cells (MaSCs) and breast cancer stem cells (BCSCs) were identified and analyzed using transplant assays (namely mammary repopulating unit (MRU) assay, mammary tumor-initiating cell (TIC) assay), which reveal their latent ability to regenerate respective normal and malignant epithelial tissues with self-renewing units displaying hierarchical cellular differentiation over multiple generations in recipient mice. "Next-generation" methods using "barcoded" normal and malignant mammary cells, with the help of next-generation sequencing (NGS) technology, have revealed hidden complexity and heterogeneous growth potential of MaSCs and BCSCs. Several single markers or combinations of markers have been reported to prospectively enrich MaSCs and BCSCs. Such markers and the extent to which they enrich for MaSCs and BCSCs activity require a critical appraisal. Also, knowledge of the functional assays and their limitations and harmonious reporting of results is a prerequisite to improve our understanding of MaSCs and BCSCs. This chapter describes evolution of the concept of MaSCs and BCSCs, and specific methodologies to investigate them.

The periodontal stem/progenitor cell inflammatory-regenerative cross talk: A new perspective.

Adult multipotent stem/progenitor cells, with remarkable regenerative potential, have been isolated from various components of the human periodontium. These multipotent stem/progenitor cells include the periodontal ligament stem/progenitor cells (PDLSCs), stem cells from the apical papilla (SCAP), the gingival mesenchymal stem/progenitor cells (G-MSCs), and the alveolar bone proper stem/progenitor cells (AB-MSCs). Whereas inflammation is regarded as the reason for tissue damage, it also remains a fundamental step of any early healing process. In performing their periodontal tissue regenerative/reparative activity, periodontal stem/progenitor cells interact with their surrounding inflammatory micro-environmental, through their expressed receptors, which could influence their fate and the outcome of any periodontal stem/progenitor cell-mediated reparative/regenerative activity. The present review discusses the current understanding about the interaction of periodontal stem/progenitor cells with their surrounding inflammatory micro-environment, elaborates on the inflammatory factors influencing their stemness, proliferation, migration/homing, differentiation, and immunomodulatory attributes, the possible underlying intracellular mechanisms, as well as their proposed relationship to the canonical and noncanonical Wnt pathways.

Long-term culture and cloning of primary human bronchial basal cells that maintain multipotent differentiation capacity and CFTR channel function.

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome-editing tools. Recently, conditional reprogramming of cells (CRC) with a Rho-associated protein kinase (ROCK) inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the life span of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial cell growth medium, instead of F medium, and 2% O2, instead of 21% O2, that extend HBEC life span while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell, and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof-of-concept, CRISPR/Cas9 genome editing and cloning were used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.